The mechanism of insulin action on gene expression is a key question in biology, with important ramifications for the treatment of diabetes and metabolic disorders. Studies supported by this grant have established a role for the O sub-family of Forkhead transcription factors in insulin regulation of gene expression. During the past funding cycle, we have demonstrated that FoxOl is the principal insulin- dependent transcription factor in the regulation of hepatic gluconeogenesis and pancreatic (3 cell mass. We have shown that: /, phosphorylation is the main mechanism by which insulin inhibits FoxOl by promoting its nuclear exclusion;ii, FoxOl expression can single-handedlyconfer insulin regulation on the expression of Glucose-6-phosphatase, the rate-limiting enzyme in glycogenolysis;Hi,FoxOl interacts with Pgcla to stimulate gluconeogenesis;iv, FoxOl links insulin signaling to Pdxl regulation of |3cell mass. We now present preliminarydata extending the gamut of FoxO functions to regulation of cell differentiation and protection against oxidative stress, while also expanding the repertoire of FoxO target genes. We demonstrate that these functions are based on two novel modes of FoxO action: acetylation-dependent 'targeting to nuclear Pml bodies, and protein/protein interactions with the Notch effector Csl. The latter observation indicates that FoxOl functions as a coactivator, and not only as a transcription factor. It also bridges together two important signalingpathways, the PI 3-kinase and the Hesl pathways. In the next five years, we will endeavor to integrate this new information in the mechanism of FoxOl action and its role in metabolic disorders. We propose to study: in Aiml, how phosphorylation- and acetylation-mediated mechanisms are integrated in vivo to determine the kineticsof FoxOl sub-cellularlocalization in physiologic conditions and disease states;in Aim 2, how acetylation-dependent sub-nucleartargeting of FoxOl regulates metabolism in liver, pancreatic (3 cells and adipocytes;and in Aim 3, how the balance between the coactivator and transcription functions of Foxol is determined. The studies will be carried out with genetic loss- and gain-of-function experiments in transgenic mice and cultured cells, using methods that have been fully implemented in the Pi's laboratory. The ultimate goal of this work is to find a therapeutic approach to modifying FoxOl function. Indeed, while FoxOl is an extremely attractive biological target to treat diabetes and metabolic diseases, it is largely intractable as a drug target. Therefore, it is hoped that by identifying mechanisms of action and interacting partners, new ways to modulate its function can be found.